Fluorescence microscopy is an invaluable tool for biologists, which provides high-resolution, high-speed, protein-specific imaging in living cells, tissues, and animals. In particular, fluorescence microscopy uses markers which absorb light and spontaneously re-emit that light at a different wavelength. The nanosecond-scale lag between absorption and spontaneous emission limits how much light a fluorescent marker emits per second, which fundamentally limits measurement speed and precision. For example, seeing individual steps of a RNA polymerase as it moves along a DNA strand requires a fluorescent marker which emits thousands of photons per millisecond, much more than typical fluorescent proteins can produce. As such, greatly increasing the brightness and photostability of fluorescent markers would enable high speed, high precision measurements which are currently impossible.
Spontaneous emission is not the only way for an excited marker to emit light after absorption. If an excited marker is illuminated with light of the proper color, it can also be “stimulated” to emit. Since the rate of stimulated emission can be much faster than spontaneous emission, stimulated emission can be several orders of magnitude brighter than spontaneous emission, an exciting possibility for improving fluorescent marker brightness. However, stimulated emission is difficult to distinguish from the stimulating light—it is the same color, the same phase, the same polarization, and, in bulk materials, goes the same direction. Noise and background from the stimulating beam is therefore difficult to reject, which negates the advantage of stimulated emission for increasing marker brightness. As such, no method to cleanly distinguish stimulated emission from the stimulating beam is currently known.